The invention relates to the field of molecular biology. In particular, the invention relates to isolated polynucleotides encoding diacylglycerol O-acyltransferase.
Oils obtained from plant seeds are important sources of fatty acids for human consumption and for use as chemical feedstocks. These fatty acids include essential fatty acids, saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. In plant seed oils, fatty acids are stored predominantly as triacylglycerols (TAGs). TAGs represent the most efficient form of stored energy in eukaryotic cells.
TAG biosynthesis occurs in the endoplasmic reticulum, in plastids, and in oil bodies, and uses sn-glycerol-3-phosphate and acyl-CoAs as its primary substrates (Stymne and Stobart, 1987; Oo and Chew, 1992; Frentzen, 1993). Biosynthesis of TAG is effected through a biochemical process generally known as the Kennedy pathway, and involves the stepwise esterification of three fatty acyl moieties to the glycerol backbone, the fatty acyl moieties being derived from acyl CoA. Each step is catalyzed by a different acyltransferase. Prior to the final acylation, the phosphate at the sn-3 position of sn-1,2-diacylglycerol phosphate (phosphatidate) is removed via the catalytic action of phosphatidate phosphatase. The final step is the acylation of sn-1,2-diacylglycerol (DAG) by diacylglycerol O-acyltransferase (DGAT; EC 2.3.1.20) to form TAG. The acylation of DAG to form TAG, catalyzed by DGAT, is the only committed step in the Kennedy pathway, and it has been suggested that DGAT may be rate limiting in plant storage lipid accumulation (Ichihara et al., 1988; Perry and Harwood, 1993; Settlage et al., 1998).
In view of its potential rate-limiting function, it has been suggested that DGAT is a potential target in the genetic modification of plant lipid biosynthesis. For instance, increased DGAT abundance in plant oilseeds could lead to increased seed oil content and improvements in the fatty acid composition of the oil. Further, because DAG is an important signaling molecule that activates protein kinase C, DGAT activity may potentially affect cellular signal transduction.
DGAT is also present in animal cells, and has been identified as perhaps being involved in: (a) intestinal fat absorption; (b) lipoprotein assembly and the regulation of plasma TAG concentrations; (c) fat storage in adipocytes; (d) energy metabolism in muscle; (e) milk production; and, (f) egg production, including the production of mammalian oocytes. Genetic manipulation of DGAT may be useful in affecting these traits. For instance, regulation of DGAT activity could have value in changing the marbling characteristics in beef, resulting in meat cuts of higher quality and commercial value.
Kamisaka et al. (1997) describe purification of DGAT from Mortierella ramanniana var. angulispora. Additionally, a few isolated DGAT coding sequences are known. Cases et al. (1998) disclose a predicted amino acid sequence of murine DGAT, and a corresponding coding sequence is disclosed in GenBank Accession No. AF078752. GenBank Accession Nos.AJ131831 (Hills et al., 1999) and AJ238008 (Zou et al., 1999) provide DGAT coding sequences of Arabidopsis thaliana. However, to the knowledge of the applicants, the prior art does not disclose Brassica napus (canola) DGAT coding sequences or polypeptides.
It appears that B. napus might contain as many as four or five different DGAT genes. Although DGAT catalyzes only a single reaction, i.e. the acylation of DAG to TAG, the chain-length of the acyl-CoA substrate may vary, as may the degree of saturation of the substrate. In B. napus, C18 acyl-CoAs are predominant, and these may be saturated (C18:0), monounsaturated (C18:1), or polyunsaturated (C18:2 or C18:3). Isolated coding sequences for DGAT having specificity for unsaturated acyl-CoAs would be useful for producing more highly unsaturated TAGs, which have been shown to have human health benefits. Canola oil has the lowest saturation level (6-7%) of the common edible oils, even lower than soya or corn oil. Hence, canola oil is valued as a healthful oil for human consumption. However, the saturation level of canola oil has been slowly increasing over time, perhaps due to narrowing of the gene pool of B. napus strains currently cultivated. The result is that soya- or corn-based oil products have become more competitive with respect to saturation level. Transformation of B. napus with polynucleotides encoding DGAT having specificity for unsaturated acyl-CoAs may be a strategy for reducing the saturation level of canola oil.
Moreover, over-expression of DGAT in B. napus or other oilseed plants, irrespective of its acyl-CoA specificity, would have the benefit of increasing TAG production in the plant. Use of native B. napus DGAT coding sequences for over-expression of DGAT in B. napus would be particularly advantageous because some of the concerns surrounding genetically modified organisms (GMOs) might be avoided. It will therefore be to value to isolate various B. napus DGAT genes.
The invention provides isolated polynucleotides (hereinafter described as xe2x80x9cDGAT polynucleotidesxe2x80x9d) which encode polypeptides having DGAT activity and which comprise amino acid sequences having at least 95% sequence identity, and more preferably 98% sequence identity, to the amino acid sequence depicted in SEQ ID NO: 2, or having at least about 95% sequence identity, and more preferably at least about 98% sequence identity, to the amino acid sequence depicted in SEQ ID NO: 4, and which have a length of at least 300 amino acid residues, preferably at least 400 amino acid residues, and even more preferably at least 500 amino acid residues. Preferably, the isolated DGAT polynucleotide encodes a full-length naturally-occurring Brassica napus DGAT. In an exemplified case, the isolated DGAT polynucleotide encodes the amino acid sequence depicted in SEQ ID NO: 2. In another exemplified case, the isolated DGAT polynucleotide encodes the amino acid sequence depicted in SEQ ID NO: 4.
The invention further provides polynucleotide constructs, vectors, and cells comprising DGAT polynucleotides.
The invention also provides isolated polypeptides having DGAT activity (hereinafter described as xe2x80x9cDGAT polypeptidesxe2x80x9d), and which are encoded by the isolated DGAT polynucleotides of the invention.
Also provided are transgenic plants, plant cells, callus, seeds, plant embryos, microspore-derived embryos, and microspores, comprising DGAT polynucleotides.
The invention also provides methods for making recombinant plants comprising DGAT polynucleotides, methods for producing DGAT, and methods for modulating DGAT activity in plants.
The invention further provides methods for producing oils, and methods for producing TAGs.
The compositions and methods of the invention are useful in a wide range of industrial, agricultural, and medical applications. In particular, the compositions and methods of the invention are useful for improving seed oil content and fatty acid composition in plants, particularly Brassica napus. Use of isolated B. napus-derived DGAT polynucleotides of the invention for the over-expression of DGAT in B. napus is particularly useful, as it may avoid at least some of the concerns over GMOs.